Our company has established a comprehensive bioactive natural products target identification technology platform, with a strong emphasis on label-free target discovery approaches. Among these, drug affinity responsive target stability (DARTS) technology has emerged as a robust, versatile, and physiologically relevant method for identifying direct protein targets of small molecules without chemical modification. DARTS plays a central role in our integrated platform, enabling efficient and unbiased target identification for natural products and other bioactive compounds.
What is DARTS?
DARTS is a label-free, proteomics-based target identification technology that exploits a fundamental property of protein–ligand interactions: ligand binding often stabilizes the target protein's structure. When a small molecule binds to its target protein, it can protect the protein from proteolytic degradation. DARTS does not require chemical labeling, immobilization, or structural modification of the compound, making it particularly suitable for natural products, which are often structurally complex and sensitive to derivatization. The technology allows researchers to identify direct binding proteins in native biological systems, such as cell lysates or tissue extracts, preserving near-physiological conditions.
Principle of DARTS
The principle of DARTS is based on differential protease sensitivity between ligand-bound and unbound proteins. Under controlled conditions, a non-specific protease partially digests proteins in a biological sample. In the absence of a ligand, proteins are more susceptible to proteolysis due to their intrinsic flexibility. However, when a small molecule binds to its target protein, the interaction often induces conformational stabilization, reducing accessibility to protease cleavage sites. As a result:
- Target proteins bound to the compound exhibit increased resistance to proteolysis
- Non-target proteins are degraded at similar rates in both treated and control samples
By comparing proteolytic patterns between compound-treated and untreated samples, stabilized protein bands can be detected and subsequently identified using mass spectrometry. This stabilization effect serves as a direct indicator of physical interaction between the compound and the protein.
Fig. 1. Schematic diagram of DARTS strategy [1].
Our DARTS-Based Technology Platform
Our company has established a comprehensive DARTS-based technology platform specifically optimized for bioactive natural products. This platform integrates experimental design, proteolysis optimization, advanced proteomics, and bioinformatics analysis to ensure high sensitivity and reproducibility. We can tailor protease selection, digestion conditions, and compound concentrations to accommodate diverse natural product classes, including alkaloids, flavonoids, terpenoids, polyketides, and peptides.
Typical Workflow of DARTS Includes:
Step 1
Native cell lysis
Cells or tissues are lysed under non-denaturing conditions to preserve protein–ligand interactions.
Step 2
Compound and control treatment
The lysate is divided into an active natural compound group and a solvent control group. The compound group is incubated with the bioactive molecule, while the control group receives solvent only.
Step 3
Protease gradient digestion
Both groups are subdivided into five to six samples and subjected to limited proteolysis using gradient concentrations of non-specific proteases, such as subtilisin, thermolysin, or Streptomyces griseus protease.
Step 4
Protein analysis
Digested samples are analyzed by SDS-PAGE or 2D-PAGE with appropriate staining to detect proteins stabilized by compound binding.
Step 5
Target identification
Differentially protected proteins are identified using gel-based or non-gel-based mass spectrometry.
Fig. 2. The typical process of DARTS [2].
Key Advantages of Our DARTS Platform Include:
- Label-free detection, avoiding compound modification
- Broad applicability to small molecules with unknown chemistry
- High specificity for direct binding proteins
- Compatibility with complex biological matrices
- Scalability, from hypothesis-driven studies to unbiased discovery
Our optimized protocols minimize false positives and maximize confidence in target identification.
Expanded Applications of Our DARTS-based Platform
Our DARTS platform not only supports target identification, but also enables a range of downstream studies, including:
- Mechanism-of-action studies of natural products
- Target deconvolution of phenotypic screening hits
- Drug repurposing and off-target profiling
- Early-stage drug discovery and lead optimization
DARTS technology represents a powerful, label-free approach for uncovering the direct molecular targets of bioactive compounds. Its unique advantages make it particularly well-suited for natural product research, where traditional target identification methods often fall short. By integrating DARTS with complementary label-free technologies, we provide our clients with high-confidence, biologically relevant target identification, accelerating the translation of bioactive natural products into validated drug candidates.
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References
- Li G., et al. Currently available strategies for target identification of bioactive natural products[J]. Frontiers in Chemistry, 2021, 9: 761609.
- Jiang X., et al. Recent advances in identifying protein targets of bioactive natural products[J]. Heliyon, 2024, 10(13).